The goal of this project is to elucidate the function of GM1 ganglioside in the nuclear membrane. The developmental expression of GM1 at this locus, which occurs during axon outgrowth, is different from that of teh plasma membrane and its functional role is different. Our study of neuroblastoma cells indicated a requirement for elevated intracellular calcium ( [CA2+]i ), and to support the physiological relevance of that finding we plan to study 2 primary neuronal cultures. The latter will also be used to test the developmental link between nuclear GM1 expression and axonal but dendrite-) outgrowth. Our primary hypothesis is that the function of nuclear GM1 is to facilitate calcium efflux from the nucleus by potentiating a sodium-calcium exchanger present in the nuclear envelope. That such an exchanger is present in this envelope is a new finding which we support with immunoblot analysis and immunoprecipitation; association of this exchanger with GM1 is indicated by Western blot analysis with cholera toxin B subunit and thin-layer chromatography. An effort will be made to isolate this exchanger protein and characterize its structure as well as its location in the inner vs outer nuclear membrane. The function of the exchanger will be investigated with a GM 1-null mutant of NG108-15 cells (NG-CR72); calcium signaling in these cless has been shown to differ significantly from wild type and the GM1- deficient cells undergo apoptosis on exposure to elevated [CA2+]i . Rescue with LIGA-20, a membrane permanent derivative of GM1 that enters the nuclear membrane, has been demonstrated and will now be applied to cerebellar granule neurons (CGN) from ganglioside knockout mice lacking GM2/GD2 synthase (GaINAc-T). The latter have been shown to resemble NG-CR72 cells in lacking calcium regulation when exposed to elevated K+, leading to apoptosis. CGN from the knockouts will be subjected to glutamate as an alternative means of elevating [CA2+]i. A stroke model will also be employed.